Aerobic fermentation process



United States Patent O 3,546,071 AEROBIC FERMENTATION PROCESS John D. Douros, Jr., Littleton, Colo., Lars A. Naslund, Roselle Park, N.J., and William J. Lahl, Marysville, Ohio, assignors to Esso Research and Engineering Company, a corporation of Delaware No Drawing. Filed Oct. 9, 1967, Ser. No. 673,925 Int. Cl. C12d 13/06 US. Cl. 19528 7 Claims ABSTRACT OF THE DISCLOSURE Thus, the present invention is broadly concerned with a biosynthesis fermentation process carried out in the presence of cellulose. More particularly, the invention is concerned with the use of oxygenated hydrocarbons par- 3,546,071 Patented Dec. 8, 1970 ever, this technique depends upon the availability of large quantities of relatively expensive carbohydrates which add significantly to the cost of the process and product.

Another recent, and even more promising, technique for biologically synthesizing food protein is by cultivating the microorganisms on petroleum substrates. This type of protein fermentation synthesis is usually conducted in an aqueous biosynthesis bath containing a hydrocarbon feed, an inocculant of the microorganism to be grown, an aqueous growth medium, oxygen, nitrogen and other indispensable nutrients. This technique allows the use of hydrocarbon feeds, which are widely available in the necessary quantities and are less expensive than carbohydrates. It is also known to use various biological catalysts in fermentation processes. The biosynthetic process of the present invention is applicable to the biosynthesis of all microorganisms which are capable of growth on oxygenated hydrocarbon substrates, particularly those oxygenated substrates derived from petroleum hydrocarbon fractions.

While the present invention is applicable to a broad scope of operable microorganisms, there are a number of microorganisms which are especially suitable for oxidized hydrocarbon assimilation. These microorganisms are tabulated hereinbelow, along with their identification numbers such as A.T.C.C. registration numbers which were secured by depositing samples with the American Type Culture Collection, 212 M. Street, Northwest, Washington 7, DC, or other designated numbers.

ice

BACTERIA Division Class Order Family Tribe genus Species Number Protophyta- Sohizomyeetes--Pseudomonadales Pseudomonadaeeae P M lz'gustr; ATCC 15522.

pseudomallei ATOC 15523.

'- orm'lla ATCC 15524.

Eubaeterialw T MlOIOCL Mz'cr cerifican ATCC 14987.

Sarcina sp. QMB1518 Natick.

l-Brevibaeteriaceae Brevibacterium--r insectiphilium ATCC 15528.

healiz' ATCC 15527.

l-Achromobacterac A" M sp. ATCC 15525.

-Corynebact:eriaeeae-- C'ellumonas galba A'ICC 15526.

iC'orynebacteriumsp. ATCC 15529.

paurometabolum ATCC 15530.

'Arthrobacter -1- simplea: ATCC 6946.

. -sp. ATOC 19140.

Actinomycetales1- Mycobacteriaceae Mycabacterium rhodchrous- ATCC 13808.

Actinomycetaceae Nocardt'a 8p.

ticularly water-soluble, oxygenated hydrocarbons as the primary source of carbon in the process. In accordance with a specific adaptation of the present invention, hydrocarbon fractions, such as petroleum liquids and gases, are oxidized, or partially oxidized, and then contacted with a microorganism under fermentation conditions in the presence of cellulose to produce high yields of protein. The process may be carried out continuously or batchwise.

The present world shortage of protein, especially lowcost animal proteins, for consumption by animals and humans is well known. In an attempt to alleviate this protein shortage, there have recently been developed several biosynthesis processes wherein biologically produced protein is provided by the growth of microorganisms on various carbon-containing substrate materials. One such technique involves growing various microorganisms, such as yeast and bacteria, on carbohydrate substrates. How- The Micrococcus cerificans (14987), which was isolated and identified by Dr. R. E. Kallio et al., Journal of Bacteriology, vol. 78, No. 3, pages 441-448 (September 1959), is particularly desirable. Further identification is as follows:

MORPHOLOGY GRAM REACTION Negative. Colonies on defined agar are small (1 mm.), circular convex, having entire edge. Colonies on nutrient agar are larger (2 to 5 mm.), raised mucoid, generally round.

Within a species there can be many different strains comprising variations and both natural and induced mutants.

The morphology and growth reaction characteristics of other organisms listed above are given in U.S. Pat. 3,308,035 issued Mar. 7, 1967 entitled, Process for Producing a High Protein Composition by Cultivating Microorganisms on an N-Aliphatic Hydrocarbon Feed, inventor John D. Douros, J r.

The growth media comprise an aqueous mineral salt medium and excess oxygen. Oxygen is supplied to the cultivation substrate medium or broth in any form capable of being assimilated readily by the inoculant microorganism. Oxygen-containing compounds may also be used to supply oxygen as long as they do not adversely affect microorganism cell growth and the conversion of the oxidized hydrocarbon feed to microorganism cells. Oxygen may be supplied as an oxygen-containing gas, such as air at atmospheric or elevated pressure or oxygen-enriched air wherein the oxygen concentration may be up to 70% to 90%. In general, between about 0.1 and about 10, preferably between about 0.8 and about 2.5 volumes per minute of air are supplied to the reactor per volume of liquid in the fermentor.

Nitrogen is essential to biological growth. The source of nitrogen may be any organic or inorganic nitrogen-containing compound which is capable of releasing nitrogen in a form suitable for metabolic utilization by the growing microorganism. Suitable organic nitrogen compounds are, for example, proteins, acid-hydrolyzed proteins, enzyme-digested proteins, amino acid, yeast extract, asparagine, and urea. Suitable inorganic nitrogen compounds are ammonia, ammonium hydroxide, nitric acid or salts thereof such as ammonium phosphate, ammonium citrate, ammonium sulfate, ammonium nitrate and ammonium acid pyrophosphate. A very convenient and satisfactory method of supplying nitrogen to the process is to employ ammonium hydroxide, ammonium phosphate or ammonium acid phosphate, which can be added as the salt per se or which can be produced in situ in the aqueous fermentation media by bubbling ammonia gas or gaseous ammonia through the broth or injecting aqueous ammonium hydroxide into the broth to which phosphoric acid was previously added, thereby forming ammonium acid phosphate. In this way the desired pH range of about 3.0 to 8.5 is maintained and the requisite nitrogen supplied. If the microorganism comprises a yeast the preferred pH is in the range of 3.0 to 7.5 such as 4.0 to 5.0. If the microorganism comprises a bacteria the desired pH is in the range of 5.0 to 8.5, such as about 7.0. Ammonium hydroxide may be supplied to the biosynthesis bath in amounts of between about 0.08 and about 0.20, preferably between about 0.1 and about 0.15, gram of nitrogen per gram of dried cells produced. This amounts to between about 0.01 and about 1.0 wt. percent, preferably between about 0.1 and about 0.15 Wt. percent, nitrogen based on the total biosynthesis bath.

In addition to the oxygen and nitrogen, it is necessary to supply requisite amounts of selected mineral nutrients in the feed medium in order to insure proper microorganism growth and maximize the assimilation of the oxidized hydrocarbon by the microorganism cells. Potassium, sodium, iron, magnesium, calcium, manganese, phosphorous, and other nutrients are included in the aqueous growth medium. These necessary materials may be supplied by any technique but are preferably supplied by their water-soluble salts.

Potassium may be supplied as potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium acetate and potassium nitrate. Iron and phosphorous may be supplied in the form of their sulfates and phosphates, such as iron sulphate and iron phosphate. Usually, most of the phosphorous is supplied as ammonium phosphates. When either ammonium phosphate or ammonium acid phosphate is used, it serves as a combined source of both nitrogen and phosphorous for the microorganism cell growth.

One satisfactory composition for the fermentation media particularly for bacteria at the outset of fermentation is as follows:

Concentration (grams per liter) Remainder to equal 100 wt. percent Cellulose is preferably in strips such as about to 2' wide and 5' to 6' long. Desirable srtips are wide and 1' long.

Other optional mineral nutrients which may be included in trace amounts include:

Concentration (milligrams per liter) hereinabove.

A very satisfactory medium is prepared as follows:

P MEDIUM Grams/liter of tap water (NH HPO 10 K HP Na SO To the above is added cc./ liter of a salt solution A prepared as follows:

Salt Solution A: Grams/ liter distilled water MgSO .7H O 40 FCSO47H2O 2 MnSO .4H O 2 N aCl 2 The foregoing P medium has a pH of 7.8. A variation of the above is one in which phosphate is supplied in the form of phosphoric acid.

The temperature of the biosynthesis bath may be varied between about C. and about 55 C. depending upon the specific microorganism being grown, but preferred temperatures when using bacteria are between about C. and about C. such as about 35 C. The pH is preferably in the range from 5.5 to 8.5 such as about 7.0.

The carbon source, preferably the sole carbon source, for the fermentation process is an oxygenated hydrocarbon other than carbohydrates, sugars, starches and cellulose. The oxygenated hydrocarbons should supply greater than about of the carbon required, preferably supply greater than about of the carbon required, preferably all of the carbon required. The oxygenated hydrocarbons may comprise alcohols, ketones, aldehydes, esters and acids, preferably those compounds containing from about 1 to 50 carbon atoms in the molecule which are secured by oxidizing a petroleum fraction. Preferred oxygenated hydrocarbons are those secured from a petroleum fraction which contains from about 2 to 20 carbon atoms in the molecule. A desirable compound is selected from alcohols containing from about 2 to carbon atoms in the molecule particularly ethyl alcohol.

Thus, the broad concept of the present invention is to utilize oxygenated materials such as oxygenated natural gas and oxygenated hydrocarbon petroleum fractions boiling up to about 900 F. and particularly those containing from about C -C carbon atoms in conjunction with cellulose strips. These compounds are selected from alcohols, esters, aldehydes, ketones and acids. Preferred oxygenated compounds are the alcohols, especially ethyl alcohol. Other desirable oxygenated compounds are, for example, isopropyl alcohol, methyl ethyl ketone, acetic acid, gly cols, =butyl alcohol, acetone, octanol, decanoic acid, dibutyl ester of sebacic acid and dimethyl ad-ipate. Suitable alcohols are methanol, ethanol, n-propanol, isopropanol, C -C primary alcohols, straight chain or branched, 2- ethyl hexanol, isoctyl alcohol, glycol, glycerine, cyclohexanol, C -C secondary alcohols from either n or isoparaflin feeds, monoethoxylates of secondary alcohols, and crotyl alcohol (unsaturated C alcohol). Suitable ketones and aldehydes are acetone/MEK (methylethyl ketone), MIBK (methylisobutyl ketone), isoborone, cyclohexanone, cyclopenta-none, stearyl aldehyde, lauryl aldehyde, acetaldehyde, and crotonaldehyde (unsaturated C aldehyde). Suitable acids are acetic acid, propionic acid, caproic acid, capric acid, lauric acid, oleic acid, stearic acid, linolelic acid, parachidic acid, toluic acid, oxalic acid, sebacic acid, terephthalic acid, phthalic acid, malonic acid, hexahydromellitic acid, and crotonic acid (unsaturated C acid). Suitable esters are methyl acetate, ethyl acetate, ethyl butyrate, ethyl stearate, glycol diacetate isopropyl valerate, dimethyl phthalate, glyceryl trioleate, and sec-C alcohol acetate; and suitable ethers are ethoxylates, diethyl ether, tetrahydrofurane, and diisopropylether.

Petroleum gases, such as methane, ethane, propane and butane, can be air oxidized to alcohols, ketones, aldehydes, ethers and acids by conventional methods, such as a rain ing solids technique or in a packed bed reactor. As mentioned, oxidized substrates are preferably derived from hydrocarbon fractions containing from about C -C car- 'bon atoms especially those having from about C -C hydrocarbon atoms. It is to be understood that other techniques and processes may be utilized for manufacturing of oxygenated material from C -C hydrocarbons.

It is also within the concept of the present invention to utilize a crude oxygenated fraction such as a crude fraction having the following composition:

MIXTURE A--OXYGENAT'ED COMPOUNDS Percent Methyl alcohol 56.6 Acetone -a. 29.0 Ethyl alcohol 8.1 Ethylacetate 2.7 Methylacetate 2.0 Dimethyl propane 0.3 t-Butylalcohol 0.3 Methylethyl keytone 0.3 Methylisopropenyl ketone 0.3 0 0.3

The cellulose is preferably cellulose acetate strips which are prepared by treating wood pulp with acetic acid, acetic anhydride and sulfuric acid as a catalyst. The cellulose is fully acetylated (three acetate groups per glucose unit) and at the same time the sulfuric acid causes degradation of the cellulose polymer so that the product contains only about 200-300 glucose units per polymer chain. At this point in the process the cellulose acetate is partially hydrolyzed by the addition of water until an average of 22.5 acetate groups per glucose unit remain. This product is a thermoplastic.

As pointed out heretofore, unexpected, desirable results are secured by the addition of the cellulose strips to the fermentation process in that the growth rate and the conversion rate of the oxygenated hydrocarbon is markedly increased.

The present invention may 'be more readily understood by the following examples illustrating the same.

EXAMPLEl Micrococcus cerificans (14987) was grown under conditions described (P medium) except cellulose strips were added in addition to the various oxygenated hydrocarborgs. The results secured are listed in the following Ta eI.

TABLE I.MICROCOCCUS CERIFICANS GROWN ON VAR- IOUS OXYGENATED HYDROCARBONS *All compounds added on an equal carbon basis (480 mmoles).

From the foregoing data it is evident that cell growth rates and oxygenated hydrocarbon conversion rates can be greatly increased by the use of cellulose strips. Cell weights are increased from 3 to 444% depending on the oxygenated hydrocarbon used. No growth is observed with cellulose and Micrococcus cerificans without an oxygenated hydrocarbon carbon source.

EXAMPLE 2 The protein content, essential amino acid index and amino acid profile of Micrococcus ceriflcans (14987) grown on various pure oxygenated hydrocarbons and harvested in Example 1 were determined using the customary analytical procedures and calculations.

The protein content (expressed as a percent) is calculated from the determined weight percent nitrogen (as determined by the Kjeldahl method) of the cells by multiplying by a factor of 6.25.

The essential amino acid index of the harvested cells is determined, using the conventional method employing egg as a basis for comparison. Egg is considered as a perfect protein having an essential amino acid index (E.A.A.) of 100.0.

In determining the amino acid profiles of the harvested cells, chromatographic analysis was used to determine all listed components with the exception of tryptophan, which was determined by microbiological assay.

The protein contents and essential amino acid indexes (E.A.A.) for the harvested cells of Micrococcus cerificans are indicated in Table 11, while the amino acid profiles are shown in Table III.

TABLE II.PROTEIN CONTENT AND E.A.A. INDEX OF MICROCOCOUS CERIFICANS ON VARIOUS OXYGEN- AIED HYDROCARBONS TABLE III.AMINO ACID PROFILE OF MICROCOCCUS CERIFICANS ON VARIOUS OXYGENATED HYDROCARBONS Grams amino acids/100 grams of protein Oxygenated hydrocarbon. Cellulose strips 1 Not determined.

2 Cetyl alcohol.

3 Ethyl alcohol.

4 Sec-butyl alcohol. 5 Oleic acid.

H Methyl alcohol.

1 Iso-propyl alcohol.

From the foregoing it is evident that cellulose strips substantially increase the growth rate of cells on an oxidized hydrocarbon. Also, the harvested cells produced possess the valuable combination of a high protein content in excess of 57 percent, a high essential amino acid index in excess of 47 percent and a nutritionally attractive amino acid profile. Thus, the present invention is especially useful in preparing human or animal feed supplements having significant protein and over-all nutritional value.

What is claimed is:

1. A continuous aerobic fermentation process for preparing a high protein product which comprises continuously supplying as a primary source of carbon an oxygenated hydrocarbon having from about 1 to 30 carbon atoms in the molecule, and continuously supplying a nonlimiting, liquid, aqueous, inorganic salt growth medium, an excess oxygen-containing gas to a vigorously stirred reactor containing said oxygenated hydrocarbon and cellulose acetate strips, aqueous medium and Micrococcus ce ificans inoculated in said reactor, temperature in said reactor being maintained in the range from C. to 47 C. and the pH of said reactor being maintained in the range from 3.0 to 8.5 and thereafter continuously harvesting the high protein product.

2. Process as defined by claim 1 wherein the liquid residence time is from about 1.5 to 4 times the minimum generation time for Micrococcus cerificans.

3. Process as defined by claim 1 in which the oxygencontaining gas supplied to the reactor is from about 0.5 to 4 volumes of air per volume reactor liquid per minute per weight percent dry cell concentration in the reactor per liquid residence time in hours.

4. Process as defined by claim 1 which comprises continuously supplying a mixture of 0.1 to 10 wt. percent of oxygenated hydrocarbons.

5. Process as defined by claim 4 wherein said oxygenated hydrocarbon is ethyl alcohol.

6. Improved aerobic microbial fermentation process for the production of a high protein content product which comprises utilizing, as a primary source of carbon, an oxygenated hydrocarbon having from about 1 to 30 carbon atoms in the molecule, conducting the fermentation process in the presence of cellulose acetate strips under aerobic conditions utilizing an aqueous inorganic salt growth medium, maintaining the temperature in the range from about 20 C. to 47 C., maintaining the pH in the range from about 3.0 to 8.5 and utilizing Micrococcus cerificans as the microorganism and thereafter harvesting the high protein product.

7. Process as defined by claim 6 wherein said oxygenated hydrocarbon is selected from the group consisting of cetyl alcohol, ethyl alcohol, secondary butyl alcohol, oleic acid, methyl alcohol and isopropyl alcohol.

References Cited UNITED STATES PATENTS 3,308,035 3/1967 Douros -428 ALVIN E. TANENHOLTZ, Primary Examiner S. RAND, Assistant Examiner US. Cl. X.R. 

